Beta carbolines are complex heterocyclic structures with specific side groups attached at various points, differentiating members of this class of compound. The skeleton ring structure of beta carbolines consists of an indole group attached to a cyclic amine (FIG. 1). This structure has numerous conjugated double bonds which impart color to the compound via light absorption and emission spectra. Upon stimulation of these compounds with ultraviolet light, strong fluorescence is produced. Quantitative analysis utilizing the ultraviolet and fluorescent spectra of beta carboline has been shown to be accurate and sensitive (Bosin and Jarvis 1985; Inoue et al. 1983).
Alternate terms for beta carbolines found variously in the current scientific literature include:
Norharmane=beta-carboline (basic parent molecule) PA0 Harman=harmane PA0 Harman=1-methyl-beta-carboline PA0 Harmine=7-methoxy-1-methyl-beta-carboline PA0 Harmalol=7-hydroxy-beta-carboline PA0 Harmalan=1-methyl-3,4-dihydro-beta-carboline PA0 Harmaline=7-methoxy-3,4-dihydro-beta-carboline PA0 Pinoline=6-methoxy-1,2,3,4-tetrahydro-beta-carboline PA0 Tetrahydroharmane=1-methyl-1,2,3,4-tetrahydro-beta-carboline PA0 Tetrahydroharmane=1-methyl-tetrahydro-beta-carboline PA0 Tetrahydroharmane=tetrahydroharman PA0 Tetrahydronorharmane=1,2,3,4-tetrahydrobeta-carboline PA0 Tetrahydronorharmane="tryptoline" PA0 Tetrahydronorharmane=tetrahydro-beta-carboline PA0 Tetrahydronorharmane=tetrahydronorharman. PA0 a. obtaining viable cells; PA0 b. treating said cells with a beta carboline; PA0 c. irradiating said treated cells with ultraviolet light; and PA0 d. observing fluorescence from said irradiated cells, said fluorescence being indicative of neurotransmitter receptor or storage sites. PA0 a. adding a quantity of a beta carboline to a mammalian tissue sample with a functioning neurotransmitter system, said beta carboline accumulating in the functioning neurotransmitter system of said sample; and PA0 b. fluorescently imaging the sample, wherein areas of concentrated fluorescence correspond to reservoirs of beta carboline accumulated by the functioning neurotransmitter system. PA0 a. obtaining from a mammal a viable tissue sample comprising a functional neurotransmitter system; PA0 b. adding a pharmacologic neurotransmitter agonist or antagonist to the tissue sample; PA0 c. adding an amount of a beta carboline which labels the functional neurotransmitter system; PA0 d. fluorescently imaging the tissue sample, wherein areas of concentrated fluorescence correspond to reservoirs of beta carboline accumulated by the neurotransmitter system; and PA0 e. comparing quantity or rate of the beta carboline fluorescent labeling to control values obtained without presence of neurotransmitter agonist or antagonist or to control values separately determined, wherein an effective level of the neurotransmitter agonist or antagonist significantly decreases areas or intensity of beta carboline fluorescent labeling. PA0 a. adding to a tissue sample from an individual, neurotransmitter agonists or antagonists which block undesired localization of beta carboline; PA0 b. interacting the sample with a proposed or standardized psychopharmacologic agent used to affect the neurotransmitter system; PA0 c. adding a quantity of a beta carboline which accumulates in the neurotransmitter system; PA0 d. fluorescently imaging the tissue sample, wherein areas of concentrated fluorescence correspond to reservoirs of beta carboline accumulated; and PA0 e. comparing beta carboline images to those obtained from a tissue sample obtained when step (b) is omitted.
As noted above these beta carbolines have individual names, i.e., harmaline, harmane, etc., indole alkaloids, harmala alkaloids, and rarely seen older terms such as tryptolines and pyridoindoles. For purposes of the present invention the term "beta carboline" is intended to include the above listed compounds and any related compounds useful as vital stains for neurological tissue.
The basic beta carboline structure bears resemblance to endogenous indoleamine neurotransmitters, namely tryptamine, serotonin, melatonin, and others. The indoleamine structure consists of an indole heterocyclic group with various amine and hydroxyl groups attached. The beta carboline molecule is also structurally similar to the norepinephrine receptor blocker yohimbine and to a lesser degree, beta carbolines resemble catecholamine neurotransmitters, e.g., epinephrine, norepinephrine, and dopamine.
Naturally occurring beta carbolines have been isolated from plants and animals. A number of alkaloid beta carboline derivatives have been extracted from plants, each with variations on the number of double bonds present on ring 3 and side groups on ring 1 (FIG. 1) (Glennon 1981). These plant extracts (harmala alkaloids) have experimentally been found to interact with neurotransmitter systems (Rommelspacher 1978). FIG. 1 also schematically shows the structure of indole; serotonin; harmol; harmine; harmalol; and harmaline.
Researchers have examined the interactions of these compounds with the serotonergic systems within the brain. Various beta carbolines have been found to antagonize serotonin receptors. (Glennon 1979). Harmaline is one beta carboline which has a high binding affinities for serotonin receptors. (Glennon 1981). The precise biochemical mechanism of serotonin antagonism by harmaline is only partly clarified. Harmaline is known to block cell membrane uptake of serotonin as well as receptor binding (Airaksinen et al. 1981). Some researchers have accounted for this by interaction of beta carbolines with an "imipramine receptor," the site at which tricyclic antidepressants bind and inhibit serotonin uptake (Airaksinen et al. 1978; Langer et al. 1984).
In addition to interactions with serotonin sites, beta carbolines are known to inhibit a number of other neurological binding sites. For instance, harmine and other related beta carbolines exhibit potent interactions with the benzodiazepine receptor (Rommelspacher et al. 1981) as well as known endogenous neurotransmitter receptors for acetylcholine, opiate, serotonin, and dopamine sites. One study has shown that the IC.sub.50 (concentration required to inhibit 50% of binding) value of Harmaline for opiate and muscarinic cholinergic sites was about four times lower than for serotonin or dopamine sites, but, in contrast, was about four times higher than found for benzodiazepine antagonism (Muller et al. 1981). Accordingly, Harmaline is a far more potent antagonist of benzodiazepine binding than it is for serotonin or dopamine.
In vivo studies of the effects of beta carbolines in animals support the in vitro findings of various selective neurological interactions. Harmine and some other related beta carbolines having effects not unlike a benzodiazepine antagonist would e.g., increased anxiety, CNS stimulation, and convulsions (Sigg et al. 1964; Gershon and Lang 1962; Fuentes and Longo 1971). Still other in vivo effects, psychotomimetic, tremorigenic or antipsychotic, support the findings that these compounds interact with cholinergic and serotonergic receptors as well. In addition to these interactions, various beta carbolines have been experimentally shown to interact with noradrenaline and tryptamine receptor sites in neurological tissue (Airaksinen et al. 1984; Given and Longenecker 1983).
Beta carbolines can variously locate and assess the activity or presence of neurotransmitter accumulation sites. The various neurons and other cells which release the above neurotransmitters have transport mechanisms which reaccumulate the neurotransmitter for the purported purpose of conservative reprocessing and self regulation. Some beta carbolines have exhibited potent competitive inhibition of this "uptake" (Sepulveda and Robinson 1974). The present inventors have observed that beta carbolines accumulate in discrete areas of tissue known to be rich in these uptake sites and that this accumulation can be monitored and accurately measured via e.g., fluorescent microscopy or fluorescent flow cytometry. This measurement of a neurotransmitter analogue would effectively allow the measurement of neurotransmitter accumulation sites.
The interaction of beta carbolines with receptor and uptake sites has been shown to be reversible and non-toxic to metabolic processes at low doses with 100% recovery following their application (Schonenweid et al. 1981, 1986). Beta carbolines also antagonize the action of monoamine oxidase A, the enzyme responsible for the breakdown of serotonin (Blackwell 1981; Burkart and Kettler 1977). Harmaline has been shown to potently inhibit this intracellular enzyme in vivo (Fuller et al. 1985).
The use of this group of compounds would allow the monitoring of a variety of neurotransmitter activities in living tissue. To measure the activity of receptor and uptake sites of a specific neurotransmitter, a beta carboline with a high specificity may be used. Alternatively, a pretreatment with various known neurotransmitter blocking agents (non-fluorescent) to block undesired interactions may also be employed. With the use of various pretreatment solutions followed by application of the beta carboline vital stain, the measurement of the varied aforementioned neurotransmitters would be possible.
The activity of serotonin, norepinephrine, acetylcholine, dopamine, opiate, tryptamine, and benzodiazepine systems play a significant role in many psychiatric disorders. For example, serotonin receptor number and uptake site activity changes in the pathological states of schizophrenia, depression, suicidal behavior, and others (Stahl et al. 1985). For patients with major affective depression, drugs which alter the uptake site activity of norepinephrine and serotonin, "tricyclic antidepressants" offer the mainstay of effective treatment. No generally effective method for the accurate assessment of these sites needed for the diagnosis and more importantly the effective management of these patients currently exists.
In an attempt to treat patients suffering from the above psychiatric disorders, physicians have administered tricyclic antidepressants and other neurologically active compounds to their patients. Tricyclic antidepressants are a class of psychopharmacological agents which interact with serotonergic and other neurotransmitter systems. It is believed that the action of the tricyclic antidepressants is to inhibit the re-uptake, and thus the metabolism of catecholeamines and indoleamines. Though the mechanism of action of this class of drugs is not completely elucidated, they are thought to induce a delayed uptake of the neurotransmitter at the postsynaptic receptor and thus the effect of the neurotransmitter. For example, all tricyclic antidepressants block the re-uptake of norepinephrine by adrenergic nerve cells. Nevertheless, each tricyclic antidepressant affects each neurotransmitter system in a distinctly different way, thus eliciting distinctly different responses in patients. For instance, imipramine slows the turnover rate of 5-hydroxytryptophan, an effect not shared by desipramine, while the turnover rate of norepinephrine is increased by the demethylated drugs nortriptyline and desipramine. The exact relationship of these effects to the action of the tricyclic antidepressants in human depression is not known. Therefore, it would be advantageous to provide a method useful in defining the mechanism of action of tricyclic antidepressants and other psychoactive compounds in order to better understand and treat patients suffering from these pathologies.
The treatment of patients with psychiatric disorders has usually been limited to the administration of psychopharmacological agents. The selection of a particular psychopharmacological agent is often based on the outward manifestations of the patient. For example, clinical rating scales for qualifying symptom complexes are available to define treatable target symptoms on the basis of clinical interviews and observations. However, because these drugs often elicit different effects in different patients, the choice of drug is often not based on anticipated therapeutic effect, but on the side effects of the particular drug and the patient's history using the drug. If a patient has responded well to a drug in the past, it is typically used again; if the patient's history does not indicate either a drug of choice or one to be avoided, clinical guidelines have been established for selecting and administering an agent. Generally, these guidelines consider the side effects of the drug, patient compliance, sedative effect of a drug, and general medical condition of the patient. Further, the choice of a particular drug is also conditioned on the physician's experience with the particular drug, a factor that often outweighs all others.
The effective use of these drugs also depends on the selection of an adequate dosage level. Typically, the tricyclic antidepressants are administered initially with a single dose at bedtime and increasing to a total daily dose by the end of the first week. If the patient shows no response in the first two weeks, the dose can be increased to a maximum allowable dosage. If the patient does not respond to one tricyclic antidepressant compound, another may be substituted. If the patient is unresponsive or unmanageable, electroconvulsive shock therapy may be necessary.
Some patients do not respond to psychopharmacological treatment and their disease may even worsen after treatment. Because "nonresponders" cannot be identified beforehand with certainty, the physician must accept the fact that there is a small subgroup of patients who do worse on medication than on no medication at all. The reported percentages of patients showing improvement with tricyclic drugs varies widely from about 32 to about 80%, depending on the criteria used for diagnosis and improvement. However, most psychiatrists report improvement in approximately 60 to 70% of depressed patients. It has been postulated that a disorder of amine metabolism exists in some depressed patients. Presumably, it is these patients who respond favorably to antidepressant drugs. A disorder of amine metabolism may also afflict some patients with mania. Accordingly, it would be advantageous to provide a method useful in identifying those patients who would respond favorably to psychopharmacological treatment, and more particularly, to provide a method useful in identifying the particular psychopharmaceutical agent to which a patient suffering from a psychiatric disorder would favorably respond.
As discussed above, the effective treatment of patients with psychopharmacological agents depends upon the dosage administered to the patient. Dosage is generally determined by the outward manifestations of the patient, the blood levels of the drug administered, or the breakdown products of neurotransmitter metabolism. Serotonergic receptor number and uptake site activity change in several psychiatric disorders. It is believed that psychopharmacological agents elicit their effect by interacting with these neurotransmitter systems. Ideal blood levels of psychopharmacological agents are often misleading, if not worthless, because of such changes. As previously discussed, a large percentage of patients are nonresponsive to psychopharmacological agents, regardless of their corresponding blood level. In the past, breakdown products of neurotransmitter metabolism have been measured in the blood, urine and cerebrospinal fluid to follow the progress of a patient's treatment. However, examining catabolites of neurotransmitters is an indirect method which may be influenced by several factors, particularly in a system modulated by psychopharmacological treatment. Thus, presently there is no method for accurately determining the dose of a psychopharmacological agent necessary to treat a patient suffering from a psychiatric disorder. Accordingly, it would be advantageous to provide a method to measure the activity of neurotransmitter uptake sites which are, in fact, the target of tricyclic antidepressant and other psychopharmacological agents. Further, it would be advantageous to provide a vital stain for neurotransmitter systems which could be utilized to determine the activity of neurotransmitter uptake sites in a patient. Thus, optimum drug dose could be predicted for individual patients depending on their unique physiology. A beta carboline used as described above would advantageously provide researchers with a tool for understanding psychiatric disorders, choosing particular psychopharmacological agents useful in the treatment of psychiatric disorders, and selecting an effective dosage of a psychopharmacological agent for a patient in need thereof. In addition, this method of assessing the activity of neurological systems may provide medicine with the first effective objective test with which to diagnose schizophrenia.
Because of the inaccessibility of brain tissue, many of the receptor and uptake sites of interest are impractical to analyze. However, lymphocytes and platelets are alternative tissue sources that contain serotonin uptake and receptor sites (Stahl et al. 1985). Human platelets are perhaps the best developed peripheral model and the most extensively studied. They are rich in serotonin receptors and uptake sites (Stahl and Meltzer 1978; Sneddon et al. 1969, 1971, 1973; paul et al. 1980, 1981a) as well as imipramine binding sites (Briley et al. 1979) (some authors have hypothesized a closely associated but separate receptor for serotonin and imipramine, a potent pharmacologic serotonin uptake inhibitor, while other authors present a single site model). Platelets, like lymphocytes, have monoamine oxidase, alpha-2 adrenergic receptors, and Na/K ATPase. In addition lymphocytes are rich in beta-adrenergic receptors. The receptor/uptake systems of platelets and of central nervous system have been suggested by others to be analogous in view of the many demonstrated similarities.
For instance, in major depressive disorders the capacity and number of serotonin uptake sites is decreased in platelets as well as in central nervous system tissue (Justice et al. 1988; Briley et al. 1980: Paul et al. 1981b; Stanley et al. 1982). Fluorescent cell sorting (Flow cytometry) is a recognized tool which, through recent developments, can measure kinetics of bound and unbound ligands in equilibrium using fluorescent labeling techniques. Lymphocytes and platelets are particularly amenable to study with the present methods. Another method, spectrofluorometry, may be used to evaluate the total amount of fluorescence given off from a solution. Though this peripheral model of neurological tissue is relatively new, it has gained wide acceptance. With the availability of fluorescent neurotransmitter analogues which can stain unfixed tissue, together with accurate methods to analyze staining, this method of investigating the nervous system should contribute significantly to the development and effective use of psychoactive drugs.